US8591921B2 - Induction of tumor hypoxia for cancer therapy - Google Patents

Induction of tumor hypoxia for cancer therapy Download PDF

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US8591921B2
US8591921B2 US12/922,658 US92265809A US8591921B2 US 8591921 B2 US8591921 B2 US 8591921B2 US 92265809 A US92265809 A US 92265809A US 8591921 B2 US8591921 B2 US 8591921B2
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tirapazamine
hypoxia
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stilbene
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Ruey-min Lee
Peck-sun Lin
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Virginia Commonwealth University
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Definitions

  • the invention generally relates to compositions and methods to increase the ability of hypoxia-activated bioreductive agents to kill tumor cells within solid tumors.
  • the invention provides methods and compositions to create local regions of hypoxia within a tumor, or within a region containing a tumor, in order to enhance activation of and tumor cell killing by hypoxia-activated bioreductive agents within the local region.
  • Tumor growth requires the development of a network of neovasculature to supply oxygen and nutrients and to remove toxic metabolites.
  • the neovasculature in tumors differ significantly from normal vasculature (1, 2).
  • Tumor neovasculature is abnormal, chaotic and inadequate in structure and function, and some data suggest that tumor vasculature may rely more on tubulin as the cytoskeletal support in contrast to both tubulin and actin as the cytoskeleton in normal tissue (3).
  • Targeting tumor vasculature has evolved into a useful strategy to develop new cancer therapeutics (4).
  • Two approaches are currently used to target tumor vessels. One is to prevent the angiogenic process by blocking angiogenic factors or their receptors in order to prevent new vessel formation.
  • VEGF vascular endothelial growth factor
  • sorafenib or sunitinib small molecular inhibitors of VEGF receptor tyrosine kinase (4-8).
  • VDAs vascular disrupting agents
  • CA4 combretastatin A4
  • ZD6126 ZD6126
  • AVE8062 Oxi4503
  • stilbene derivatives 9-13
  • hypoxia-Induced Factor (IIIF) 1- ⁇ 14, 15
  • hypoxia-Induced Factor (IIIF) 1- ⁇ 14, 15
  • Nitric oxide (NO) another factor that is produced in tumor cells during hypoxia, induces vasodialation and thus also improves tumor blood supply (16, 17). NO is also tightly linked to angiogenesis (18, 19). These compensatory mechanisms can thus result in drug resistance against anti-angiogenic agents and VDAs.
  • VDAs are currently in phase II trials for various solid tumors but none have received FDA approval thus far.
  • One new strategy is to combine anti-angiogenic agents with VDAs. This is based on the observation that VDAs induce elevation of VEGF, which subsequently mobilizes bone marrow endothelial progenitor cells into circulation and is thus responsible for repair of damaged tumor vessels (20). Using an inhibitor of the VEGF pathway could possibly block the mobilization and enhance therapeutic effects (11, 20), but this strategy has not been proved in clinical setting.
  • Tirapazamine (SR4233; 3-amino-1,2,4-benotriazine-1,4-di-N-oxide) is a bioreductive agent that works exclusively in hypoxic environments and has also been tested as an anticancer agent (21).
  • Tirapazamine is activated by cytochrome P450 reductase by a one-electron reaction, thereby generating nitroxide radicals. In the absence of oxygen, nitroxide radicals induce single- and double-strand breaks in DNA to cause cell death. Because of this property, tirapazamine exhibits 15-200-fold greater toxicity to hypoxic cells compared to well-oxygenated cells. This agent has also been shown to be a radiation sensitizer and to act synergistically with platinum compounds in cancer therapy regimens (22, 23).
  • a postulated mechanism of action of tirapazamine is shown in the following figure (24-28).
  • One electron reduction of tirapazamine proceeds through reductive activation by enzymes including cytochrome P-450, NADH-cytochrome P-450 reductase and other flavo- or metalloproteins.
  • the product for the single electron transfer reaction is a free radical (I or II), which can be oxidized by oxygen to yield a superoxide and the parent drug tirapazamine.
  • the free radical (I or II) can acquire a second electron through a hydrogen abstraction reaction intermediate (III) to form a stable mono-N-oxide (SR4317).
  • liver is the most important organ for the metabolism of tirapazamine.
  • Costa et al. examined the toxicity of tirapazamine in cultured rat hepatocytes that were transiently incubated in a hypoxic environment at oxygen concentrations of 1, 2, 4, 10 and 20% (29).
  • the dose response curve of tirapazamine to monolayer hepatocytes shifted significantly to the left, meaning more susceptible to death, as the oxygen concentration decreased (P ⁇ 0.05).
  • the concentration of tirapazamine that caused 50% cell death over 2 h at 4% oxygen was more than 10-fold less than that required at 10% or 20% oxygen concentration.
  • the concentration of tirapazamine required to induce 50% cell death at 2% oxygen was 15-fold less than that required to induce same degree of cell death at 20% oxygen.
  • tirapazamine Significant development of tirapazamine has been done both pre-clinically and clinically. Animal studies of tirapazamine showed that its potential side effects include bone marrow toxicity, necrosis of the olfactory nerve, and retinal degeneration (21). A phase I clinical study of tirapazamine by intravenous administration every three weeks showed a maximum tolerated dose (MTD) of 390 mg/m 2 , and dose limiting toxicities include reversible ototoxicity and transient visual abnormality when the dose was above 330 mg/m 2 (30, 31). Other non-specific toxicities included muscle cramps, nausea, vomiting, and diarrhea. Grade 1 thrombocytopenia was noted in one patient receiving 450 mg/m 2 and no leucopenia was noted in any patient (30).
  • MTD maximum tolerated dose
  • the invention is based on the development of methods and compositions that enhance the anti-tumor activity of hypoxia-activated bioreductive agents (HABAs), while reducing or minimizing the side effects that can occur as a result of systemic administration of such agents.
  • HABAs hypoxia-activated bioreductive agents
  • the administration strategies of the invention involve inducing hypoxia at localized regions where it is desirable to activate a HABA, for example, within a tumor or in an area that contains a tumor.
  • a HABA is also present in the localized hypoxic region, the HABA is activated and exerts its killing effect on cells in the region (e.g. tumor cells), without having a deleterious systemic effect on the organism.
  • the bioreductive agent is administered prior to or concomitant with direct mechanical occlusion of a vessel, i.e. embolization.
  • Embolization can be confined in an isolated, targeted region, resulting in the development of hypoxia in the isolated, targeted region and hence activation of the HABA.
  • a HABA is administered to a tumor in combination with one or more hypoxia-inducing agents such as vascular disrupting agents (VDAs) and anti-angiogenic agents (AAAs).
  • VDAs vascular disrupting agents
  • AAAs anti-angiogenic agents
  • Such agents may also be considered as “chemical embolization” agents as this approach uses VDAs and AAAs (chemical agents) to achieve the purpose of vascular occlusion, in contrast to direct physical occlusion of a vessel using mechanical embolization.
  • VDAs and AAAs chemical agents
  • standard embolizing agents e.g. Lipiodol
  • VDAs and AAAs agents that may be delivered without embolization
  • hyperoxia-inducing agents are referred to as “hypoxia-inducing agents”.
  • hypoxia-inducing agents may, optionally, also be delivered during a mechanical embolization procedure.
  • mechanical embolization local or systemic delivery of agents such as VDAs and AAAs together with a HABA, selectively creates a localized hypoxic environment in a tumor in which the HABA is activated and kills the surrounding tumor cells, but the HABA will remain in the inactive prodrug form in the non-hypoxic region systemically.
  • these methodologies are combined, e.g.
  • AAAs and/or VDAs, and/or direct mechanical embolization are administered or carried out together with provision of a HABA, so that the selective hypoxic region induced by these approaches can lead to activation of the HABA.
  • the overall effect of these inventive techniques is the rapid, efficacious and synergistic destruction of tumor cells in the localized area, without extensive systemic involvement and deleterious side effects.
  • Such methods include embolization, vascular disrupting agents or anti-angiogenic agents individually or in combination.
  • the process is combined with administration of a hypoxia-activated bioreductive agent, which is activated exclusively in the hypoxic region to induce tumor cell death. Systemic toxicities will also be minimized to gain the maximal benefit.
  • the invention thus provides a method of selectively killing tumor cells in an animal.
  • the method comprises the steps of: 1) providing the animal with a hypoxia-activated bioreductive agent; and 2) locally forming, within a tumor or a defined area containing one or more tumors, a region of hypoxia of 10% or lower oxygen.
  • the hypoxia activated bioreductive agent becomes activated for killing tumor cells at the region of hypoxia within the tumor or defined area.
  • the step of locally forming the region of hypoxia is achieved by providing the animal with one or more of vascular disrupting agents and anti-angiogenic agents (i.e.
  • the step of providing the animal with one or more of vascular disrupting agents and anti-angiogenic agents is performed systemically.
  • the step of providing the animal with one or more of vascular disrupting agents and anti-angiogenic agents is performed locally at the region.
  • the step of locally forming the region of hypoxia is performed after the step of providing.
  • the step of locally forming the region of hypoxia may be performed simultaneously with the step of providing.
  • the locally forming step provides one or more vascular disrupting agents selected from combretastatin, combretastatin derivatives, (5S)-5-(acetylamino)-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-3-yl dihydrogenphosphate (ZD6126), DMXAA (5,6-dimethylxanthenone-4-acetic acid, (N-[2-[4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide) (E7010 or ABT-751), stilbene derivatives such as cis-3,4′,5-trimethoxy-3′-aminostilbene (stilbene 5c) and cis-3,4′,5-trimethoxy-3′-hydroxystilbene (stilbene 6c) or their derivatives, and a prodrug morpholino-carbamate derivatives, and a prod
  • the locally forming step provides one or more anti-angiogenic agents selected from bevacizumab, sorafenib, sunitinib, aflibercept, IMC-1C11, vatalanib (PTK-87), N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide (AMG 706), 3-(4-Bromo-2,6-difluoro-benzyl oxy)-5-[3-(4-pyrrolidin-1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide (CP-547,632), pazopanib (GW-786034), N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N′-(2-fluoro-5-methylphenyl
  • the hypoxia activated bioreductive agent is tirapazamine
  • the locally forming step includes the step of providing cis-3,4′,5-trimethoxy-3′-aminostilbene (stilbene 5c).
  • the locally forming step includes providing bevacizumab.
  • the step of locally forming may include the steps of administering one or more vascular disrupting agents followed by administering one or more anti-angiogenic agents.
  • the step of locally forming the region of hypoxia is performed by embolization.
  • Embolization may include the step of administering one or more embolizing agents.
  • the step of providing is performed prior to said step of locally forming. In other embodiments, the step of providing is performed simultaneously with the step of locally forming.
  • the hypoxia activated bioreductive agents may be tirapazamine, banoxantrone (AQ4N), porfiromycin, apaziquone (EO9), 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), dinitrobenzamide mustard derivative (such as PR 104) or 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC 709257).
  • the region is located in the liver of said animal. In some embodiments of the invention, a level of oxygen in said region of hypoxia is 5% or lower. In some embodiments of the invention, the step of providing is performed locally, whereas in other embodiments, the step of providing is performed systemically.
  • the invention also comprises a composition or kit for selectively killing tumor cells in an animal.
  • the composition or kit comprises 1) tirapazamine, and 2) one or more of anti-angiogenic agents and vascular disrupting agents.
  • the one or more of anti-angiogenic agents and vascular disrupting agents are selected from bevacizumab, sorfenib, sunitinib aflibercept, IMC-IC11, vatalanib, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide (AMG 706), 3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin-1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide (CP-547,632), pazopanib (GW-786034), N
  • the one or more of anti-angiogenic agents and vascular disrupting agents includes cis-3,4′,5-trimethoxy-3′-aminostilbene (stilbene 5c). In another embodiment, the one or more of anti-angiogenic agents and vascular disrupting agents includes bevacizumab
  • FIGS. 1A and B Study of tumor perfusion using accumulation of gadolinium at 30 min after gadolinium injection.
  • the left panels show the T1-weighted images before injection of gadolinium.
  • the tight panels show images at 30 min after injection of gadolinium.
  • the upper panels show gadolinium enhancement in tumor and kidney before mice were treated with stilbene 5c.
  • FIG. 2 Stilbene 5c treatment decreases microvascular density in tumor but not in normal organs.
  • Nude mice with UCI tumor xenograft were treated with 10% DMSO or stilbene 5c at 50 mg/kg intraperitoneally. Mice were sacrificed at 4 hours after injection, and various organs and tumor were harvested for fixation and standard hematoxylin and eosin (H & E) staining. Immunohistochemical staining of each section was performed with anti-CD34 antibody to quantify the microvascular density. In the original immunohistochemical stained sections, brown staining indicated a positive signal for CD34 staining. (A) Shown are the black and white pictures in 200 ⁇ fold magnification.
  • FIG. 3 Tumor blood flow and oxygen content in tumor.
  • Oxygen and blood flow were monitored by OxyFlo and OxyLite system (Optronix, Oxford, UK). Mice were anesthetized with isoflurane and a triple sensor that records temperature, oxygen and blood flow was inserted into tumor and left for 1 hour for recording. Shown are the results of three mice with similar size subcutaneous tumors derived from UCI-101 ovarian cancer cells. Top panels are oxygen level and lower panels are tumor blood flow. Timing of St5c injection was marked.
  • FIG. 4 Efficacy of stilbene 5c in vivo and enhanced effect by bevacizumab.
  • Nude mice were injected with UCI-101 cells subcutaneously and mice are treated with stilbene 5c at 20 mg/kg/day Monday-Friday with or without bevacizumab 10 mg/kg twice a week. Tumor volume was calculated by the long and short axes. Each group contains 8 mice and the average tumor volumes and standard deviations were plotted against days.
  • FIG. 5A-D Synergism between tirapazamine and stilbene 5c.
  • Nude mice with UCI101 tumor xenograft were treated with vehicle ( 5 A, control), tirapazamine alone ( 5 B), stilbene 5c (50 mg/kg) alone ( 5 C) or tirapazamine (60 mg/kg) followed by stilbene 5c (50 mg/kg) ( 5 D) to induce tumor hypoxia.
  • Mice were sacrificed three days later and tumors were harvested for H&E section. Shown are the low power views of tumor sections. The portion of tumor that stained darker is viable, whereas the portion in lighter color is the necrotic area. Please note that in the tumor section at the right lower panel in the combination group. The majority of the tumor is necrotic, and only a small portion of tumor in the center and peripheral rim remain viable. The central viable portion is proximal to the branches of larger vessels.
  • FIG. 6 Synergistic effect of stilbene 5c and tirapazamine.
  • Nude mice with subcutaneous tumors were treated with (1) normal saline control, (2) stilbene 5c at 50 mg/kg, (3) tirapazamine 60 mg/kg, (4) combination of stilbene 5c and tirapazamine at days 8, 10, and 12 by intraperitoneal injection. Mice were then sacrificed at day 14 due to weight loss. The long and short axes of tumor were measured to calculate tumor size and plotted against days. Each treatment group contains 6 mice.
  • FIG. 7 Combination of tirapazamine and bevacizumab.
  • Nude mice with subcutaneous tumors were treated with (1) normal saline control, (2) tirapazamine 60 mg/kg at days 8, 10 and 12, (3) combination of tirapazamine at days 8, 10 and 12 and bevacizumab (10 mg/kg) at days 8 and 11. Mice were then sacrificed at day 14. The long and short axes of tumors were measured to calculate tumor size and plotted against days. Each treatment group contains 6 mice.
  • the present invention provides new, synergistic treatment combinations to kill solid tumor cancer cells, and methods of using the new treatment combinations to treat malignant or cancerous solid tumors.
  • the new combinations locally enhance the anti-tumor activity of hypoxia-activated bioreductive agents (HABAs) such as tirapazamine, while reducing or minimizing the side effects that heretofore have occurred as a result of the systemic administration of HABAs.
  • HABAs hypoxia-activated bioreductive agents
  • the bioreductive agent is an inactive prodrug; conversion to an active form occurs under conditions of low oxygen (hypoxia).
  • active forms of the bioreductive agents are advantageously confined to a desired area of action, e.g. within a tumor or in a circumscribed, defined area that includes one or more tumors.
  • VDAs and/or anti-angiogenic agents are administered locally or systemically to create a localized hypoxic region within which a co-administered HABA is activated.
  • VDAs and/or anti-angiogenic agents are administered locally or systemically to create a localized hypoxic region within which a co-administered HABA is activated.
  • embolization plus one or more hypoxia-inducing agents the overall effect being targeted, localized provision of an activated bioreductive agent, and efficacious killing of tumor cells within or at the targeted site without the side effects usually caused by systemic exposure to bioreductive agents.
  • This enhancement may also permit the use of lower doses of the agents while maintaining an adequate and efficacious level of tumor cell killing, thereby further decreasing toxicity.
  • region of hypoxia we mean that the level of oxygen within the region is at least lower or less than about 10%, and preferably lower or less than about 5%.
  • the level of oxygen in the hypoxic region may be about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.
  • an oxygen level of about 10% or lower, and preferably about 5% or lower is sufficient to activate hypoxia-activated bioreductive agents such as tirapazamine to a level that is at least 10-fold more active than the prodrug form.
  • oxygen measurements may be expressed in “mm Hg”, wherein, for example, 10% O 2 is equal to 76 mmHg and 1% O 2 is equal to 7.6 mmHg.
  • hypoxia-activated bioreductive agents include but are not limited to tirapazamine, banoxantrone (AQ4N), porfiromycin, apaziquone (EO9), 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), dinitrobenzamide mustard derivative (such as PR 104) and 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC 709257) (37-39).
  • the increase in activity is at least about 10 fold, or greater, and the increase may be much higher e.g. from about 20-200 fold, or from about 50 to 200 fold, or 100 to 200 fold.
  • the classic and most clinically advanced bioreductive agent tirapazamine is utilized in the practice of the invention.
  • Tirapazamine has thus far failed in clinical development.
  • phase I and phase II trials tirapazamine by itself was tolerated in doses up to 390 mg/m 2 .
  • a detailed examination of the failed phase III clinical trials shows that tirapazamine was combined with chemotherapeutic agents such as carboplatin and paclitaxel (36).
  • the trial also adopted the standard approach of increasing the dose of tirapazamine to a level close to its maximally tolerated dose, and hence created the problems of non-specific toxicity such as neutropenia, hypotension, fatigue, neuropathy, hearing loss etc.
  • the systemic distribution of the chemotherapy-tirapazamine combination explains the enhanced systemic side effects of this combination.
  • the present invention provides novel approaches to improve the efficacy of tirapazamine while minimizing adverse side effects that have up to now been observed.
  • symbolization we mean a localized therapy used in a tumor or a region containing tumor that is supplied by an identifiable arterial branch, for example, a hepatic artery that supplies a hepatocellular carcinoma, by injecting materials (Lipiodol, gelfoam, blood clot, etc.) to induce occlusion of the branch of the artery supplying the region containing the tumor so that the tumor cells cannot obtain adequate blood flow and die.
  • materials Lipiodol, gelfoam, blood clot, etc.
  • normal liver is supplied by dual vessels, the hepatic artery and the portal vein, and thus allows the occlusion of the hepatic artery or a branch of it without the consequence of significant damage to normal liver.
  • This procedure is generally performed by interventional radiologists, who place a catheter from the femoral artery in the groin and advance the tip of the catheter to the branch of the hepatic artery that supplies the tumor under fluoroscope X-ray guidance.
  • embolizing agents such as Lipiodol or gelfoam, are injected to occlude the branch.
  • This procedure is a standard loco-regional therapy for the treatment of hepatocellular carcinoma (40-43).
  • a hypoxia-activated bioreductive agent such as tirapazamine is combined with embolization for the treatment of tumors in localized areas.
  • HABA hypoxia-activated bioreductive agent
  • embolization provides a hypoxic tumor environment to enhance the effect of tirapazamine.
  • administration of tirapazamine with embolization limits the distribution of tirapazamine to the region that is supplied by the embolized vessel.
  • embolization-induced hypoxic effect is limited to the tissue that is supplied by the vessel that is embolized, no systemic hypoxia is induced and systemic toxicity due to activated tirapazamine is thus avoided.
  • tirapazamine is mixed or dissolved in an agent commonly used for standard embolization such as Lipiodol.
  • an agent commonly used for standard embolization such as Lipiodol.
  • tirapazamine is trapped within the tumor along with the embolizing agent such as Lipiodol.
  • the embolizing agent such as Lipiodol.
  • This unique combination thus has the significant advantage of fully exploiting the cell-killing capability of tirapazamine and completely eliminates the problem of systemic toxicity observed in the previous clinical studies (36), in which tirapazamine was administered intravenously along with conventional chemotherapy.
  • Lipiodol is the most frequently used embolizing agent.
  • embolizing agents that may be used in the practice of the invention include but are not limited to gelform, blood clots, nanoparticles or any clinically proven mechanical agent that can achieve the purpose of vascular occlusion.
  • the administration of embolizing agents and a hypoxia-activated bioreductive agent (HABA) can be carried out in any suitable manner.
  • the HABA may be administered prior to administration of the embolizing agent (e.g. from about 1-120 minutes before), and subsequent administration of the embolizing agent “traps” the HABA in the region.
  • the two agents may be administered together (e.g. using a preparation that includes the two agents in a mixture).
  • the dosage of HABA that is administered will be in the range of from about 1 mg to about 200 mg (e.g. of tirapazamine), and preferably from about 5 to about 50 mg, for a patient being treated by this method; and the dose of embolizing agent that will be administered will be in the range of from about 5-40 ml, and preferably from about 20-30 ml of e.g. Lipiodol.
  • Sufficient embolizing agent is administered to achieve complete occlusion of the intended branch of the vessel under fluoroscope X-ray examination, to ensure the creation of a hypoxic region or condition in the embolized area.
  • Administration of the embolizing agent is usually carried out by intra-arterial injection. Alternatively, embolization may be carried out by other means such as particular beads to induce occlusion.
  • Types of cancers that can be treated by this method include any that occur in positions within the body which can be isolated via embolization, for example, hepatocellular (liver) carcinoma, cholangiocarcinoma, and metastatic cancer from colon or other gastrointestinal organs.
  • embolization for example, hepatocellular (liver) carcinoma, cholangiocarcinoma, and metastatic cancer from colon or other gastrointestinal organs.
  • This strategy can be used for any cancer which can be treated by embolization, or for any tumor that is located within an area of the body that can be embolized without unduly harming the patient, such as sarcoma of the limbs.
  • this technique is used in the treatment of hepatocellular carcinoma as this type of cancer is routinely treated by embolization.
  • Chemotherapy is also commonly administered, called chemoembolization, in which chemotherapy is administered simultaneously with embolizing agents to trap chemotherapy agents exclusively in the embolized region to minimize the systemic toxicity and enhance therapeutic benefit.
  • the mechanical embolization in not permanent, and the duration of vascular occlusion can be controlled by the proper selection of embolizing agents, permitting the activated HABA to work for a period of time to execute the effect of tumor killing, and then to be inactivated by an influx of oxygen into the area when the blood flow in the region recovers.
  • the effect of mechanical embolization plus administration of a HABA is synergistic with respect to tumor cell killing, in that the level of tumor cell killing that occurs with this combination therapy is greater than would be expected, based on when each method is used alone, i.e. the effect is not merely additive.
  • tirapazamine with embolization or chemoembolization for the treatment of, for example, hepatocellular carcinoma.
  • tirapazamine is particularly useful for hepatocellular carcinoma or primary liver cancer when it is administered by intrahepatic artery injection along with embolization.
  • P450 is abundant in liver.
  • tirapazamine works under hypoxic conditions, which are induced by embolization of the feeding hepatic artery. Tirapazamine is trapped in the liver and is then slowly released into the hepatocellular carcinoma.
  • VDAs Vascular Disrupting Agents
  • a hypoxia-activated bioreductive agent is used in conjunction with a VDA that induces hypoxia.
  • VDAs that may be used in the practice of the invention include but are not limited to: the combretastatin derivatives (9), (5S)-5-(acetylamino)-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-3-yl dihydrogenphosphate (ZD6126) (44), DMXAA (5,6-dimethylxanthenone-4-acetic acid (9), (N-[2-[4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide) (E7010 or ABT-751) (45), stilbene derivatives such as cis-3,4′,5-trimethoxy-3′-aminostilbene (stilbene 5c) and cis-3,4′,5-trimethoxy-3
  • VDAs may be considered a type of chemical embolization, which is to use a chemical agent to achieve the same goal of embolization selectively in a tumor containing region in contrast to the direct occlusion of a vessel in standard embolization described above.
  • Combinations of VDAs and hypoxia-activated bioreductive agents such as tirapazamine are surprisingly more effective than would be predicted based on the activity of either agent alone in the treatment of solid tumor malignancies, i.e.
  • VDAs given after tirapazamine allow activation of tirapazamine and increase subsequent tumor cell killing by at least 10 fold or higher, compared to the level of tumor cell killing by either agent alone.
  • This strategy may also be combined with embolization described earlier to be even more effective in inducing tumor hypoxia and tirapazamine activation.
  • a “synergistic” interaction or effect we mean that the effect of administering two (or more) agents or treatment modalities together is greater than the simple additive effect that would be expected if each agent exerted its effect independently. In other words, the agents interact in some manner that increases the total effect that is observed beyond what would be expected.
  • the administration of a tirapazamine alone to a solid tumor typically results in a level of tumor cell death of about 10-20%.
  • the administration of a VDA alone to a solid tumor typically results in a level of tumor cell death of about 10-20% as well.
  • the level of tumor cell killing is nearly 70-80%, which is greater than the simple arithmetic sum of the two levels achieved by tirapazamine alone and the VDA alone. If the effect was additive, a maximum of about 20-40% would be predicted. The level of 70-80% is thus at least about 2-4 fold higher than the maximum that would be expected if no synergism was observed.
  • synergy is observed when an HABA is administered in conjunction with the creation of localized hypoxic regions by 1) embolism, 2) administration of an AAA, or 3) administration of a VDA, or some combination of two or more of these three.
  • the level of synergy is generally at least about 2-5 fold higher (e.g. 2, 3, 4, or 5 fold higher) than would be predicted, and may be even greater (e.g. 6-10 fold or more).
  • Example 5 Representative levels of synergy that are observed are shown in Example 5 ( FIG. 5 ), in which either tirapazamine or stilbene 5c, a VDA, induces 10-20% tumor necrosis alone; whereas combination of tirapazamine and stilbene 5c induces tumor necrosis increases to 70-80%.
  • the distribution of necrosis is also mainly in the center of the tumor, which is consistent with the notion that tumor necrosis is due to suppression of tumor blood flow and induction of hypoxia, as the tumor in the peripheral region can obtain some oxygen support by diffusion from the surrounding normal tissue.
  • the dose of HABA that will be administered will be in the range of from about 100 to about 300 mg/m 2 if tirapazamine is administered systemically, and preferably from about 150 to about 250 mg/m 2
  • the dose of VDA that will be administered will be in the range of from about 10 mg/m 2 to about 100 mg/m 2 , and preferably from about 50 to about 100 mg/m 2 depending on the dose proven to be effective in suppressing tumor blood flow in the clinical trials.
  • the amount of VDA that is administered is sufficient to cause the level of oxygen in the region to decrease to below about 10%, or preferably below about 5%, a level of oxygen that increases the activity of HABAs by at least 10 fold.
  • Methods of administering VDAs include but are not limited to intravenous, intraperitoneally, intramuscular, subcutaneous, intra-arterial, direct intratumor injection and oral administration.
  • one or more HABAs are combined with AAAs such as, for example, a vascular endothelial growth factor (VEGF) monoclonal antibody (such as bevacizumab) or inhibitors of VEGF receptor tyrosine kinase (such as sorafenib or sunitinib).
  • VEGF vascular endothelial growth factor
  • Co-administration causes synergism between the effects of AAAs and the HABA by prolonging the duration of tumor hypoxia, and further enhances the therapeutic effect of the HABA.
  • AAAs that may be used in the practice of the invention include but are not limited to: bevacizumab, sorafenib, sunitinib, aflibercept, IMC-1C11, vatalanib (PTK-87), N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide (AMG 706), 3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin-1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide (CP-547,632), pazopanib (GW-786034), N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N′-(2-fluoro-5-methylphenyl)urea (ABT
  • the dose of HABA that will be administered will be in the range of from range of from about 100 to about 300 mg/m 2 if tirapazamine is administered systemically, and preferably from about 150 to about 250 mg/m 2
  • the dose of AAA that will be administered will be in the range of from about 5 to 15 mg/kg for bevacizumab, and about 200-400 mg orally twice a day for sorafenib.
  • the dosage may vary depending on potency of the agent used.
  • the amount of AAA that is administered is sufficient to cause the level of oxygen in the region to decrease to below about 10%, and preferably below about 5%, i.e. to create a hypoxic region.
  • AAA The application of AAA to induce tumor hypoxia in combination with HABAs is characterized by two critical factors.
  • the first is the half-life of the AAAs.
  • Monoclonal antibodies such as bevacizumab have a half-life of over 7 days and work by neutralization of the angiogenic factor VEGF.
  • VEGF angiogenic factor
  • the deprivation of VEGF eventually prevents new vessel formation in tumors and results in tumor hypoxia due to the consumption of oxygen within the tumor (48-50).
  • Small molecular compounds such as sorafenib and sunitinib have half lives of less than 24 hours and work by directly inhibiting the kinase activity of VEGF receptors. Therefore the timing of induction of hypoxia by AAAs can be difficult to control even when the drug half life is known.
  • AAAs can also induce temporary normalization of tumor vasculature (51-53) and actually improve tumor blood flow and oxygen level in the tumor. This theory was used to explain the rationale of combining AAAs with radiation, in which adequate oxygenation is essential for tumor killing by radiation whereas hypoxia compromises the radiation effect (51). This effect of temporary normalization of tumor vasculature by AAAs is counterintuitive to the desired hypoxic environment for activation of tirapazamine or other HABAs.
  • the invention thus proposes to combine VDAs and AAAs as an approach to induced tumor hypoxia.
  • the rationale is to use VDAs to induce immediate suppression of tumor blood flow to induce hypoxia and activation of HABAs.
  • the tumor will develop compensatory hypoxic responses, such as production of VEGF or other angiogenic factors to mobilize endothelial progenitor cells from bone marrow to repair the damaged tumor vasculature (20).
  • the components of the combination tumor therapies described herein include one or more anti-angiogenic agents (AAAs), and one or more vascular disrupting agents (VDAs), and a hypoxia-activated bio-reductive agent (HABA).
  • AAAs anti-angiogenic agents
  • VDAs vascular disrupting agents
  • HABA hypoxia-activated bio-reductive agent
  • the combination of AAAs and VDAs induce prolonged hypoxia in tumor cells when administered together, and display some efficacy in killing tumor cells on their own, but these agents administered alone have been relatively ineffective in treating solid tumors.
  • their activity is significantly enhanced in a synergistic (non-additive) manner when they are administered as described herein in combination with hypoxia-activated bioreductive agents (HABAs), which are also known anticancer agents.
  • HABAs hypoxia-activated bioreductive agents
  • the methods described herein may be considered to be methods of enhancing the anticancer activity of AAAs and/or VDAs by co-administering a HABA, or methods of enhancing the anticancer activity of HABAs by co-administering AAAs and/or VDAs.
  • the administration of the agents described herein may be carried out by any suitable means known to those of skill in the art, with the caveat that the activity of the hypoxia-inducing agent must be as nearly as possible confined to the area that is targeted for treatment, i.e. to a tumor or tumors, or to a region of the body that contains one or more tumors and can be isolated from adjacent areas.
  • VDAs have the capability of preferentially suppressing tumor blood flow without significant compromise of normal circulation (11) and thus may be administered systemically.
  • the HABA may be administered either locally or systemically, as its activation will be largely confined to the regions of hypoxia within tumors created by the action of the VDA.
  • administration of VDAs and HABAs is confined largely to a targeted area, i.e. administration is local, such as when combined with embolization.
  • administration is local, such as when combined with embolization.
  • This can be accomplished by locally administering the agents e.g. by intra-arterial injection into the vascular branch supplying a tumor through a catheter placed by interventional radiologists.
  • Such local administration may be carried out by administering individual agents one at a time in close succession as described herein, or by administering a single preparation that contains a mixture of the agents.
  • the administration of AAAs is typically systemic either orally or by intravenous injection after embolization due to the fact that AAAs are used to suppress the compensatory effect, which induce a whole body reaction, of tumor hypoxia induced by VDAs.
  • all agents including the hypoxia-inducing agent (e.g. an AAA and/or VDA) and the HABA, are administered systemically, since activation of the HABA will occur only in the localized hypoxic areas in tumors due to the preferential tumor vascular effect of VDAs.
  • Methods of systemic administration include but are not limited to oral administration, intravenous, intraperitoneal, intramuscular, subcutaneous, or intra-arterial administration, inhalation, etc.
  • the agents e.g. HABA; or VDA and HABA, with or without an AAA
  • the hypoxia-inducing agent may be administered either after the HABAs, or together with the HABAs, so long as administration results in sequestering the agents within the targeted region.
  • AAAs are given by systemic administration as the purpose of AAAs is to block the systemic compensatory mechanism.
  • the second approach is to give all three agents systemically as VDAs can specifically induce suppression of tumor vessels even given by systemic intravenous injection. This approach will be suitable for patients who are not candidates for embolization.
  • embolization is not a standard approach to treat these tumors, and all three agents (HABA, VDAs and AAAs) are typically given systemically by oral, intravenous, intraperitoneal, or subcutaneous route, usually with the sequence of HABAs first, then VDAs and than AAAs afterwards. In this way, HABAs will be distributed to tumors, and then VDAs lead to shutdown of tumor blood flow to induce hypoxia.
  • HABAs will be distributed to tumors, and then VDAs lead to shutdown of tumor blood flow to induce hypoxia.
  • local administration of these agents is also contemplated.
  • the effect of AAAs is much slower and AAAs can be administered afterwards to suppress the compensatory response induced by hypoxia.
  • compositions described herein are generally suitable for administration to a mammalian patient, and are thus, for example, physiologically compatible.
  • Such compositions include substantially purified forms of the agents, and a pharmacologically or physiologically suitable (compatible) carrier.
  • the preparation of such compositions is generally known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified.
  • the active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof.
  • the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added.
  • the composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration, e.g. the agents may be attached to a matrix in order to provide localized delivery. The final amount of each agent in the formulations may vary.
  • the amount will be from about 1-99%.
  • the compositions may contain only one agent, or a mixture of agents (i.e. only HABA or AAA or VDA or an embolizing agent, or any combination of two or more of these).
  • more than one of each type of agent may be administered in a composition, e.g. one HABA may be administered with two or more VDAs or two of more AAAs, or two or more HABAs may be administered together, etc.
  • compositions and/or kits comprising the compositions that include at least one hypoxia-activated bioreductive agent and at least one of a hypoxia-inducing vascular disrupting agent and an anti-angiogenic agent, plus a physiologically acceptable carrier.
  • compositions comprising at least one hypoxia-activated bioreductive agent and an embolizing agent used during mechanical embolization, e.g. Lipiodol, and, optionally, one or more VDAs and/or AAAs, are also provided.
  • the types of cancer that may be treated by the methods of the invention include but are not limited to cancers in which solid tumors (solid malignancies) develop, e.g. hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, colorectal cancer, anal cancer, lung cancer including small cell or non-small cell lung cancer, breast cancer, prostate cancer, ovarian cancer, testicular cancer, germ cell tumor, renal cell carcinoma, neuroendocrine cancer, gastric cancer, esophageal cancer, head and neck cancer, skin cancer including squamous cell carcinoma or melanoma, soft tissue and osteogenic sarcoma, thyroid cancer, thymoma, bladder cancer, cervical cancer, uterus cancer, central nervous tumor, Hodgkin and non-Hodgkin lymphoma. Both primary and metastatic tumors may be treated by the methods disclosed herein.
  • the methods of the invention are generally intended to treat mammals, especially humans, but that need not always be the case. Veterinary applications are also contemplated.
  • other treatment modalities may be combined with the methods of the invention, e.g. surgical removal of tumors or portions of tumors, various chemotherapy regimens, and other treatments of side effects such as treatments for nausea, appetite stimulants, vitamins, etc.
  • Tumor cell lines used in this study include UCI-101 ovarian cancer cells and Hep3B hepatocellular carcinoma cells.
  • Cells are grown in IMEM and DMEM, respectively, supplemented with 10% fetal bovine serum, glutamine, and penicillium/streptomycin in 5% CO 2 humidified environment.
  • IMEM and DMEM fetal bovine serum
  • glutamine glutamine
  • penicillium/streptomycin in 5% CO 2 humidified environment.
  • cells were harvested with 1% trypsin and washed with phosphate-buffered saline three times before injected subcutaneously into nude mice.
  • 2 ⁇ 10 6 tumor cells were injected in the back subcutaneously. Nude mice were purchased from NCI Development Therapeutic Program as described.
  • Tumor size was monitored by long and short axes (a and b, respectively) by a caliber and tumor volume was calculated by the formula ab 2 /2.
  • mice with tumor xenografts were anesthetized with 1% isoflurane and mixed oxygen.
  • DEC-MRI was performed with experimental Magnetic Resonance (MR) system (Bruker, Biospec 2.35 T/40 cm) that is dedicated to small animal imaging.
  • Jugular vein was dissected to place an IV catheter for contrast injection in acute phase study.
  • the first batch of mice was injected with 50 ⁇ L of gadolinium (OmniScan) through the jugular catheter and MRI images were collected every second to investigate the initial rate of increase of MRI signals immediately after injection. After the initial rate is established, subsequent MRI studies focused on the sustained increase in gadolinium signals, which also provide qualitative and quantitative information for tissue perfusion.
  • 20 ⁇ L of gadolinium was injected directly into tail veins. Mice were transferred into the tunnel of MRI machine within 1 minute after injection. MRI images were collected every minute for 30 min.
  • mice After mice were sacrificed, major organs and tumors were dissected and fixed in 10% formalin. Tissues were embedded in paraffin and sections were stained with H & E, and with antibody against CD34 for quantification of microvascular density. The scoring of microvascular density was based on the methods described by Weidner et al. by counting the CD34-positive signals in a photo frame of 200 ⁇ magnification using Nikon ECLIPSE E800M microscope equipped with a Diagnostic Instruments Spot RT CCD camera.
  • Tumor xenografts were subjected to oxygen and blood flow study when tumor size reached 8-10 mm in largest diameter. Tumor was punctured with 25 G needle to make a tunnel for insertion of sensor probe. The tip of the sensor probe was left in the center of the tumor.
  • Tumor oxygen level and blood flow were determined by OxyLite 2000 dual channel monitoring system with “bare-fibre” type sensor probe, which is designed for measuring tissue oxygen, temperature and blood flow using laser Doppler technique (Optonix, oxford, UK). The measurement was done in a real time basis (100 measurements per second) and the oxygen level and tumor blood flow were recording continuously.
  • each mouse was recorded first without treatment for at least 20 min before the mice was injected intraperitoneally with the tested VDA cis-3,4′,5-trimethoxy-3′-aminostilbene (stilbene 5c) at 50 mg/kg. The tumor blood flow and oxygen level was then recorded for another 20 min after treatment.
  • VDA cis-3,4′,5-trimethoxy-3′-aminostilbene (stilbene 5c) at 50 mg/kg.
  • the tumor blood flow and oxygen level was then recorded for another 20 min after treatment.
  • Stilbene 5c was investigated to examine whether it can suppress tumor blood flow and eventually lead to tumor hypoxia.
  • a rapid kinetic study showed that after 10 min of rapid increase the gadolinium signals reached plateau and persisted for at least 30 min without washing out (11).
  • Mice were first imaged without contrast to obtain a baseline ( FIG. 1 , left upper panel). The section was obtained at the center of the tumor. Kidney in the same section was used as an internal organ control from tumor in the same mice. After injected with 20 ⁇ l of gadolinium (OmniScan) via the tail vein, mice were analyzed with a rapid sequence MRI every minute for total of 30 min.
  • gadolinium OmniScan
  • mice Both tumor and kidney exhibited enhancement of MRI signals after injection of gadolinium, which represented vascular perfusion of tumor and kidney ( FIG. 1 right upper panel). Mice were then left for at least 24 hours to let gadolinium washed out. Same mice were treated with 50 mg/kg stilbene 5c by intraperitoneal injection on the second or third day. Four hours after injection of stilbene 5c, mice were imaged again before and after gadolinium injection by the same protocol and compared with the previous pair of images before stilbene treatment. In the baseline image before gadolinium injection, the T1-weighted image had a slight increase of MRI signals ( FIG. 1 , left lower panel) compared with the baseline image of untreated mice.
  • Tumor blood flow and tumor oxygen level are dependent on the location of the probe in the tumor. The more peripheral the location of the probe is, the higher the blood flow and oxygen level will be. The center portion will have lower blood flow and hence more hypoxia in general. It is very difficult to compare different mice due to individual variation and the location of tumor in which the sensor probe is placed for the study.
  • the most reliable way to interpret the result is to use the pre-treatment blood flow and oxygen level as its own control to compare with the post-treatment result since the sensor probe was kept in the same position to eliminate spatial variation. All mice were thus recorded for at least 20 min to record the pre-treatment baseline. In the baseline study, it was observed that there is a significant temporal variation as shown in FIG. 3 .
  • the baseline oxygenation and tumor blood flow show a synchronized fluctuated baseline with time.
  • the oxygen level increases correspondingly.
  • stilbene 5c was injected followed by continuous monitoring for another 20-30 min to examine the effect of stilbene in tumor oxygenation and blood flow in real time. It was noted that different tumors had very different tumor blood flow and oxygenation level as shown in the three mice here ( FIG. 3 ).
  • Mouse #1 had a big fluctuation in baseline in a cyclic pattern with blood flow between 600-1100 and oxygen level between 15-40 mm Hg. Note that the first 10 min results were not taken into consideration due to adjustment of the trauma from probe insertion.
  • Mouse #2 has a tumor blood flow between 200-300 and oxygen levels have two plateaus. One plateau is at 50-60 mm Hg and then dropped to 20-30 mm Hg at the subsequent plateau. Mouse #3 has a very high tumor blood flow between 2000-3000 and oxygen levels at 35-55 mm Hg range.
  • stilbene 5c 50 mg/kg
  • all three mice exhibited a dramatic decrease in tumor oxygen level down to less than 10 mm Hg before the recording stopped.
  • Tumor blood flow decreased to less than 25% of the level before stilbene treatment in mice #1 and 3.
  • the tumor blood flow change in mouse #2 was significant in the beginning but subsequently recovered in some degree though still lower than baseline blood flow level.
  • FIG. 5B Previously it was known that tirapazamine alone given at 70 mg/kg has no effect in tumor growth suppression, hence tirapazamine alone group ( FIG. 5B ) was not expected to have any effect.
  • the control tumor ( FIG. 5A ) and tumor treated with stilbene 5c ( FIG. 5C ) have very small areas of tumor necrosis.
  • the combination group in which tirapazamine was given 60 min before stilbene 5c which allows distribution of tirapazamine into the tumor first, followed by induction of tumor hypoxia by stilbene 5c, we observed a dramatic increase in the size of the area of necrosis in the tumor ( FIG. 5D ).
  • necrosis area is mainly in the center of tumor with sparing of a peripheral rim, suggesting that the necrosis is due to the effect of hypoxia, since the peripheral portion of the tumor can obtain blood and oxygen supply by diffusion from the surrounding normal tissue.
  • This finding supports our theory that combination of tirapazamine followed by stilbene 5c to induce tumor hypoxia can have a synergistic effect and enhance the therapeutic efficacy of either agent.
  • the size of tumor of the groups treated with stilbene 5c or tirapazamine showed a decrease to about 50% in average compared with the control, whereas the group treated with stilbene 5c and tirapazamine was further suppressed to 27% of the control. This result suggests that stilbene 5c and tirapazamine are at least additive and could be even synergistic.
  • the anti-angiogenic agent bevacizumab first induces vascular normalization in tumor (51-53). Therefore, the tumor oxygen level may even improve, instead of being suppressed, due to vascular normalization and could not enhance the therapeutic benefit of tirapazamine. Utilization of anti-angiogenic agent bevacizumab with tirapazamine will need to combine with stilbene VDA, which was confirmed to be more effective in induce tumor growth suppression.
  • One possible way to use the three drug combination is to use tirapazamine and stilbene vascular disrupting agent in the initial setting followed by a maintenance therapy with bevacizumab.

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US10159676B2 (en) 2008-04-10 2018-12-25 Virginia Commonwealth University Induction of tumor hypoxia for cancer therapy
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US10076556B2 (en) 2015-01-29 2018-09-18 Oxyrase, Inc. Methods for inhibiting tumor growth
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